Liver disease caused by infection with Hepatitis C Virus (HCV) is a leading cause of death in HIV-infected persons, and is of particular importance among injection drug users (IDUs). Several cofactors worsen HCV progression, including HIV, age, and enhanced HCV replication. Since HIV and aging impact on each other and on HCV replication, demonstrating their independent contributions to liver disease progression has been challenging up till now. This proposal is conceived to investigate the role and mechanism(s) underlying liver disease progression in HCV-infected IDUs using innovative and complementary tools in the clinic and lab. In the first aim, the effect of HIV on aging in the liver will be studied using structured longitudinal measurements of liver stiffness in a cohort of HIV-infected and - uninfected persons, and especially on the effect of suppressive antiretroviral therapy (ART) on fibrosis progression rates. Nested mechanistic studies are planned to understand how HIV contributes to the aging liver. The second aim, focused on HCV replication, leverages advances in tissue imaging and specimen collection to study single hepatocytes from archived HCV-infected human biopsies. Absent a representative model of human HCV infection, molecular signatures of HCV- infected hepatocytes will be compared within the same person to those of uninfected hepatocytes, identifying potential therapeutic targets for HCV control. The third aim is centered on understanding HCV replication in the context of HIV co-infected persons by characterizing the transition from HIV viremia to HIV control with antiretroviral therapy (ART). HIV/HCV co-infected persons who had one liver biopsy prior to ART and one after >12 weeks of ART with HIV suppression will be studied using single-cell laser capture microdissection to define the clustered decline in HCV-infected hepatocytes in the same people. The proposed studies will define key pathways of liver disease progression in HCV-infected IDUs, with particular focus on persons with HIV co-infection. The overall long-term public health impact of a successful proposal is to a) attenuate the progression of liver disease in IDUs; b) to identify relevant molecular targets for HCV control; and c) to characterize immune control of HCV in HIV/HCV co-infection.